Novel Inhibitors of SARS-CoV-2 RNA Identified through Virtual Screening

We currently lack antivirals for most human viruses. In a quest for new molecules, focusing on viral RNA, instead of viral proteins, can represent a promising strategy. In this study, new inhibitors were identified starting from a published crystal structure of the tertiary SARS-CoV-2 RNA involved in the −1 programmed ribosomal frameshift. The pseudoknot structure was refined, and a virtual screening was performed using the repository of binders to the nucleic acid library, taking into consideration RNA flexibility. Hit compounds were validated against the wild-type virus and with a dual-luciferase assay measuring the frameshift efficiency. Several active molecules were identified. Our study reveals new inhibitors of SARS-CoV-2 but also highlights the feasibility of targeting RNA starting from virtual screening, a strategy that could be broadly applied to drug development.


■ INTRODUCTION
For most known viruses, antivirals are still lacking, leading to morbidity and mortality, particularly among the immunocompromised and the elderly.Current methods target either viral or host proteins.Unfortunately, some proteins are undruggable, and inhibiting host proteins can lead to toxicity.Nowadays, a promising strategy is to target viral RNA (reviewed by Mathez and Cagno 1 ).Tertiary RNA structures conserved among viral families, and distinct from those in human RNA, can be targeted, resulting in small molecules with broad activity and reduced off-targets. 2n late 2019, the discovery of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiated efforts to develop vaccines and treatments aimed at managing the pandemic and reducing associated mortality.An interesting RNA target of SARS-CoV-2 is its −1 programmed ribosomal frameshift (−1PRF) element. 3,4The virus employs −1PRF to regulate the expression of viral proteins crucial for its replication such as the RNA-dependent RNA polymerase.SARS-CoV-2 has two overlapping open reading frames (ORFs) in its genome, denoted as ORF1a and ORF1b.The ORF1a ends with a stop codon.However, through −1PRF, the stop codon can be bypassed, allowing for the translation of the entire ORF1ab and enabling the synthesis of the replication machinery encoded within ORF1b. 1,3,5It is estimated that a −1PRF occurs in 45−69% of the translation events. 6The elements required in vitro for −1PRF include a specific slippery sequence on the viral RNA in conjunction with the formation of a tertiary RNA structure.−10 Since this arrangement is uncommon in humans, the risk of off-target effects is reduced, and targeting this RNA structure holds a significant interest. 1everal small molecules affecting −1PRF of SARS-CoV-2 have been previously identified, demonstrating the feasibility of the approach.Specifically, merafloxacin, 3,11,12 a fluoroquinolone, and geneticin, 12 an aminoglycoside, showed specific inhibition of the −1PRF of SARS-CoV-2.Geneticin, in our work, was shown to inhibit various variants of concern, and its effectiveness was maintained in human-derived respiratory tissues.Furthermore, through molecular docking, the putative binding site of geneticin in the frameshifting element was identified and validated via mutagenesis.Notably, a combination of three mutations in the pseudoknot structure, aimed to occlude the binding site, led to decreased inhibitory activity of the frameshift mediated by both merafloxacin and geneticin.
However, fluoroquinolones and aminoglycosides are antibiotics associated with toxicity in humans, 13−17 thus hindering their development as antivirals.To understand the requirements for −1PRF inhibitors devoid of toxicity, new compounds targeting this mechanism must be identified.
In this study, a new crystal structure of the tertiary viral RNA structure involved in the −1PRF of SARS-CoV-2 7 was refined by molecular dynamics.Based on the binding site identified by our previous study on geneticin's activity, a virtual screening on different structures was performed using an experimentally verified library of compounds targeting RNA.Selected molecules were used against an omicron variant of SARS-CoV-2 and in a dual-luciferase assay to confirm the inhibition of −1PRF element of the virus leading to the discovery of novel SARS-CoV-2 inhibitors.
Omicron (BA.1)SARS-CoV-2 virus was isolated from a clinical sample from the University Hospital of Lausanne using the method of Mathez and Cagno. 18ompounds.Compounds used were purchased from ChemBridge except for piperaquine (Sigma-Aldrich), merafloxacin (MedChemExpress), and geneticin (Promega).Geneticin was dissolved in water, and all other molecules were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich).The first three digits of the commercial ID of ChemBridge or the commercial name are used in the text and the figures.
Refinement of Pseudoknot.Crystal structures of the pseudoknot (PDB 7LYJ and 7MKY 7 ) were chosen.7MKY was repaired due to a missing nucleobase utilizing 7LYJ as a template.−24 Topology was generated using Amber ff99bsc0χOL3 force field, 25−30 and tip3p water was used.A cubic box was filled with NaCl with a concentration of 15 mM and a neutral charge.Energy minimization was carried out for 50,000 steps, with the temperature and pressure equilibrium 100 and 10 ps, respectively.Molecular dynamics were then conducted for 500 ns at 300 K and 1 bar.Positions were recorded at every 100 ps.The trajectory was then centered.Clustering was performed by using the GROMOS method with a cutoff of 0.15 nm.Trajectory and clustered structures were visualized using VMD version 1.94a55. 31irtual Screening.Virtual screening was conducted on Maestro Schrodinger version 2023-2. 32The repository of binders to nucleic acids (ROBIN) library 33 was prepared using LigPrep by generating possible states at pH 7.0 ± 2 with Epik 7 and with OPLS4 force field.Tautomeric forms and chiral centers specified in the input structures were preserved, while for newly generated chiral centers (e.g., quaternary nitrogen atoms on asymmetric pyrrolidine), all possible configurations were included.Overall, LigPrep enumeration resulted in 3932 structures out of the 2003 input structures.Four clusters were chosen as the input structures.The grids were generated on each cluster using a box of 20 and 30 Å as inner and outer boxes, respectively, centered on the centroid of specified residues (G3, U4, G5, U6, U29, A57, and U58).The virtual screening workflow was then independently applied to each grid in the prepared library.After the high-throughput virtual screening, 25% of the best drugs were kept for the subsequent docking steps.After SP and XP docking, 50% of the best compounds were retained.The final poses were then refined by Prime MMGBSA.Compounds binding at least three structures were visually inspected for the final selection.
Antiviral Activity.The antiviral activity of selected compounds was evaluated with a protocol similar to the one described previously. 12,18Vero E6 cells (10 5 cells in a 24-well plate) were infected with 100 plaque-forming units of SARS-CoV-2.After 1 h of incubation, the inoculum was removed, and an overlay of 0.6% Avicel gp3515 (Selectchemie AG) and 2.5% FBS in DMEM containing serial dilutions of the drug was added.Three days later, cells were fixed with 4% formaldehyde (Sigma-Aldrich) followed by staining with crystal violet (Sigma-Aldrich).Plaques were manually counted.
Dual Luciferase.The frameshift efficiency was assessed using a dual-luciferase assay as described previously. 3,12Vero E6 cells (10 4 cells in a 96-well plate) were treated with a single nontoxic dose of the compound in 10% FBS DMEM.Wildtype or in-frame control plasmids were transfected using Lipofectamine 3000 (Invitrogen).The following day, cells were washed with phosphate-buffered saline (PBS) and passively lysed.The activity of firefly and renilla luciferase was assessed with a Dual-Luciferase Reporter Assay System kit (Promega) in a white plate using a luminometer (TriStar LB 941, Berthold Technologies).Frameshift efficiency was calculated as done by Bhatt et al. 3 Cell Viability.Following the protocol of Varricchio and Mathez et al., 12 compounds were added to Vero E6 cells in serial dilution in 2.5% FBS DMEM.Three days later, cells were washed with DMEM and a final concentration of 0.5 mg/mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich) in DMEM was added for 3 h at 37 °C.Cells were lysed with DMSO, and absorbance was read at 570 nm (BioTek Epoch 2, Agilent).
Molecular Dynamics with Ligand.Molecular dynamics of the SARS-CoV-2 pseudoknot structure with ligand was conducted following the procedure mentioned above for the refinement of the structure.Ligand topology was generated using the ACPYPE server 34,35 and was added to the topology of SARS-CoV-2 RNA.RNA and the ligand were restrained simultaneously for the temperature and pressure equilibrium.
Docking of 194 Analogues.Compound 782 was prepared following the same procedure as that for the preparation of the ROBIN, as described above.The analogue was docked using compound 194 as the center of the grid on cluster A. An XP docking protocol was used followed by Prime MMGBSA refinement.Results were visualized and analyzed in MOE 2022.02. 36tatistics.Biological experiments were performed in duplicate or quadruplicate for the dual-luciferase assay and at least two independent experiments.Results are shown as mean and standard error of the mean (SEM).The effective concentration inhibiting 50% (EC 50 ) and the concentration reducing cell viability by 50% (CC 50 ) values were calculated by regression analysis using the program GraphPad Prism version 10.1.2to fit a variable slope sigmoidal dose−response curve as described by Mathez and Cagno. 18One-way analysis of variance (ANOVA) followed by multiple comparison analysis was used as a statistical test to compare grouped analysis.

Journal of Chemical Information and Modeling
screening, this structure underwent refinement through a 500 ns molecular dynamics simulation using GROMACS (Figure S1).Following the simulation, the various frames were clustered.Four representative clusters were selected to take into account RNA flexibility at the binding site (Figure 1).
Selection of Small Molecules Binding SARS-CoV-2 Pseudoknot.Potential sites on the pseudoknot of SARS-CoV-2 were identified using Site Finder of MOE.Two sites were in close proximity and involved nucleotides previously discovered in the binding site identified in our prior study with geneticin. 12These two potential sites were merged into a single binding site for a virtual screening.In the ROBIN library, 33 compounds previously shown to bind RNA experimentally and not DNA, and having drug-like properties, were docked against each selected cluster.Molecules binding to at least 3 clusters were visually inspected (21 molecules).One molecule was excluded due to its instability in water.Based on their interactions on the predicted pose and their chemical diversity, six commercially available molecules were purchased.
Antiviral Evaluation of Selected Compounds against SARS-CoV-2.The selected compounds were evaluated for toxicity on Vero E6 cells measuring their viability after 3 days of exposure (Table 1, Figure S2).Piperaquine exhibited high toxicity with a CC 50 of 5.66 μM.Compounds 188 and 229 (compound numbering refers to the first three digits of the compound ID of the ChemBridge library) were found to have a CC 50 of 80.6 and 93.03 μM, respectively.Antiviral activities of the selected compounds were subsequently evaluated against an Omicron variant of SARS-CoV-2 previously isolated from a clinical sample (Table 1).
Compounds 188, 194, and 229 showed EC 50 values of 17.16, 28.92, and 26.82 μM, respectively.To verify the specificity of the activity on the −1PRF of SARS-CoV-2, a dual-luciferase assay was performed.In this assay, the minimal frameshifting element of SARS-CoV-2 is cloned between two luciferases.The upstream luciferase is constitutively expressed while the downstream one is expressed only upon frameshifting.As a control, an in-frame construct was used where both luciferases are expressed.Additionally, two positive controls, merafloxacin and geneticin previously shown to reduce frameshifting, were included in the assay.With this method, only compound 194 showed a significant reduction in frameshift efficiency, while compound 229 showed a similar reduction but was not significant (Figure 2A,B).

Analysis and Optimization of Compound 194.
Compound 194 from our first set showed the most potent antiviral activity, displaying a reduction of frameshift efficiency and lower cytotoxicity.Consequently, it underwent further analysis.Starting from the initial binding pose postvirtual screening, a 500 ns molecular dynamic of the pseudoknot and compound 194 was performed (Figure S3).The ligand maintained its position and appeared to interact with several nucleotides such as G2, G3, and U4 through hydrogen bonds (Figure 3).G3 was previously identified as a main interactor from the binding pose postvirtual screening.
Commercially available analogues of compound 194 were purchased.Their cytotoxicity, antiviral activity, and frameshift efficiency were assessed (Figure 2).While the CC 50 for all compounds exceeded 100 μM (Figure S2), one of the analogues, compound 782, showed a more potent antiviral activity (EC 50 4.07 μM) and a significant and improved reduction of frameshift efficiency compared to 194; furthermore, the inhibition was shown to be dose-dependent Figure 1.Selected cluster for virtual screening.Four representative clusters, derived from clustering using a cutoff of 0.15 nm of the 500 ns molecular dynamics of PDB 7LYJ and 7MKY, were selected for the virtual screening.The surface area represents specified residues previously found to be in geneticin's pocket.The same residues were used to define the grid for the virtual screening.(Figure S4).Compound 782 was docked on cluster A by using compound 194 as the center of the grid.The additional protonated nitrogen showed interaction with the SARS-CoV-2 pseudoknot (Figure S5).Other analogues showed inhibitory activity against SARS-CoV-2 but without reducing frameshift except compound 269, which showed a nonsignificant reduction of frameshift efficiency.Compound 325 in contrast did not inhibit wild-type virus.
■ DISCUSSION SARS-CoV-2 relies on a programmed ribosomal frameshift to regulate the expression of viral proteins crucial for replication.By disrupting this key process, small molecules can impede the life cycle of the virus.To this end, the antiviral activity of molecules acting on the frameshift stimulating element responsible for the −1PRF of SARS-CoV-2 was evaluated.
During infection, in proximity of the ribosome translating the end of ORF1a, a pseudoknot structure is believed to be involved in the −1PRF mechanism. 3,7,10This structure was recently crystallized with high resolution and therefore represents the most accurate model obtained. 7This structure was refined by molecular dynamics, and the resulting trajectory was clustered (Figures 1 and S1).A virtual screening was performed on rigid RNA structures with RNA flexibility considered by docking the library of molecules independently on different structures.This screening was validated by assessing the antiviral activity against the Omicron variant of SARS-CoV-2 and the inhibitory effect on the frameshift efficiency with a dual-luciferase assay (Table 1, Figure 2).Piperaquine, an antimalarial drug previously shown to be active against SARS-CoV-2, 37 was among the molecules identified.However, due to its high toxicity (Figure S2) and a small selective index (Table 1), piperaquine was not further retained for analysis.Nonetheless, more promising inhibitors were discovered.Notably, compound 194 showed an EC 50 of 28.92 μM and significantly reduced the frameshift efficiency by 23% (Table 1, Figure 2).Importantly, this molecule bound to all clusters in the initial virtual screening.Following molecular dynamics simulation, the interaction of 194 with the frameshifting element was confirmed (Figures 3 and S3).
To explore the potential enhancement of the original molecule's activity and understand the functional groups required for inhibition, analogues of 194 were evaluated.Compound 782 showed an EC 50 of 4.07 μM and significantly reduced the frameshift efficiency by 38% (Figures 2 and S4).Notably, the inhibition of this compound on the wild-type virus is more potent than previously reported inhibitors, such as merafloxacin and geneticin. 12lthough the complete structural relationship with antiviral activity is not yet understood, intriguing observations were observed following minor chemical modifications of compound 194, suggesting potential selectivity against the SARS-CoV-2 pseudoknot (Figure 2).Compound 325, substituting an azacycloheptane with a morpholine, exhibited a loss of antiviral activity against SARS-CoV-2.This loss was corroborated by compound 178, which despite harboring an additional modification with a 5-chloro-1,3-benzodioxole instead of a 2,3dihydro-6-methyl-1,4-benzodioxin, also experienced a decrease in potency.Compound 269, undergoing the same modification, retained activity against the wild-type virus albeit with reduced potency and remained active in the dual-luciferase assay with a reduction in frameshift, though not statistically significant.Conversely, replacing the azacycloheptane in compound 782 with a methylpiperazine improved potency and had a favorable impact on the frameshift reduction.Protonation of the added nitrogen might facilitate additional interactions with the RNA backbone, as evidenced by docking the compound onto SARS-CoV-2 cluster A (Figure S5), thereby potentially explaining the enhanced antiviral activity.This is also supported by the results obtained when docking morpholine analogue 325.This compound, likely due to the presence of the oxygen atom instead of the basic NMe group, might not be able to interact efficiently with the RNA phosphate groups.This is reflected in the docking results, where 325 shows a much lower score than that of 782.It should be noted that all possible ionization states at pH 7 ± 2 were generated for each structure, including asymmetric protonation on the pyrrolidine ring, using Epik 7 (Schrodinger suite).Subsequently, all of the generated structures were docked into the RNA.The results shown in Figure S5 represent the best-scored protonated combination of 782.Although this fully protonated form might not represent the most abundant species in solution (calculated pK a values for 782 are shown in Figure S6), it is possible that this state is present in a productive concentration when binding to the RNA, as observed, for example, with polyamines binding to nucleic acids. 38t is important to acknowledge the limitations of our study linked to the use of the −1PRF crystal structure as a starting point for the virtual screening.The pseudoknot structure was identified several times by structural analysis 7−9 even in complex with a ribosome 3 or by simulation showing that this structure is the only viable conformation when frameshift occurs. 10Therefore, it is a valuable target.However, this region of the SARS-CoV-2 RNA is reported to have multiple possible conformations.By assessing the frameshifting element structure in infected cells, 39−41 the pseudoknot structure was not observed, possibly due to long-range interactions with other viral RNA segments.Therefore, during infection, small molecules targeting the frameshifting element might encounter other structures for which they might have a low affinity.Applying virtual screening on the different models could expand the inhibitors of SARS-CoV-2 by altering −1PRF by different mechanisms.
Another limitation is linked to the absence of RNA modifications in the starting model.−45 These modifications could potentially alter the structure or alter the binding interactions.
Furthermore, in our study, the initial compounds were selected to bind to at least three clusters.Previous studies, aimed at identifying antivirals targeting RNA, selected molecules binding single clusters from a larger set of structures. 46,47By performing a similar protocol, the initial set of molecules could be expanded by selecting additional potential inhibitors.
Our research has unveiled novel inhibitors of SARS-CoV-2 altering the −1PRF mechanism of the virus, giving insights into the requirements for inhibiting SARS-CoV-2 RNA with druglike compounds.Importantly, to the best of our knowledge, our study identified the most potent −1PRF SARS-CoV-2 inhibitor to date and laid the groundwork for a broader approach aimed at targeting viral RNA using small molecules.

■ ASSOCIATED CONTENT Data Availability Statement
The data underlying this study are openly available in Zenodo at 10.5281/zenodo.11098463.

Figure 2 .
Figure 2. Frameshift efficiency and antiviral activity of compound 194 analogues.(A) Analogues of 194 are represented.The first three digits of the ID number of the molecule (in bold) are used in the text and figures.(B) Dual-luciferase assay was conducted on Vero E6 cells.Frameshift efficiency was evaluated in the presence of inhibitors identified after the virtual screening and analogues of compound 194.Merafloxacin and geneticin were used as positive controls.(C) Antiviral activity of analogues of compound 194 was evaluated against an Omicron variant of SARS-CoV-2 on Vero E6 cells.EC 50 : effective concentration inhibiting 50% of the virus.*P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Figure 3 .
Figure 3. Binding pose of compound 194.(A, B) Binding pose of 194 on cluster A after the virtual screening.(C, D) Binding pose of 194 on cluster A after molecular dynamics of 500 ns represented by the most populated clusters after clustering with a cutoff of 0.15 nm.(A, C) Site view and (B, D) ligand interaction maps were done with MOE.

Table 1 .
Antiviral Activity against SARS-CoV-2 and Cytotoxicity of Selected Molecules after Virtual Screening a a Identification number used in ROBIN library (in bold the name used in the text); EC 50 , effective concentration inhibiting 50% of the virus; CC 50 , concentration reducing cell viability by 50%; SI, selective index.